Focus on Nitric Oxide Homeostasis: Direct and Indirect Enzymatic Regulation of Protein Denitrosation Reactions in Plants

Abstract

Protein cysteines (Cys) undergo a multitude of different reactive oxygen species (ROS), reactive sulfur species (RSS), and/or reactive nitrogen species (RNS)-derived modifications. S-nitrosation (also referred to as nitrosylation), the addition of a nitric oxide (NO) group to reactive Cys thiols, can alter protein stability and activity and can result in changes of protein subcellular localization. Although it is clear that this nitrosative posttranslational modification (PTM) regulates multiple signal transduction pathways in plants, the enzymatic systems that catalyze the reverse S-denitrosation reaction are poorly understood. This review provides an overview of the biochemistry and regulation of nitro-oxidative modifications of protein Cys residues with a focus on NO production and S-nitrosation. In addition, the importance and recent advances in defining enzymatic systems proposed to be involved in regulating S-denitrosation are addressed, specifically cytosolic thioredoxins (TRX) and the newly identified aldo-keto reductases (AKR).

Keywords: Arabidopsis thaliana; S-Nitrosoglutathione reductase (GSNOR); S-nitrosation; aldo-keto reductases (AKRs); glutaredoxins (GRXs); posttranslational modifications; reactive nitrogen species; thioredoxins (TRXs).

Figure 1

Mechanisms of ROS, RSS (a) and RNS (b) dependent posttranslational modifications of protein Cys residues. See text for details.

Figure 2

Chromosomal location, gene structure and phylogenetic relationships of h-type thioredoxins from Arabidopsis. (a) Chromosomal location of h-type TRXs from Arabidopsis. (b) Intron-exon structure of representative gene models. Exons are represented by black bars and introns by folded black lines, while 5′- and 3′ UTR regions are white boxes. Lines and bars to scale and represent total sequence length. ATG; start codon, TGA/TAA; stop codon, WCG/PPC; active site. Scale bars represent 100 nucleotides. (c) Maximum likelihood phylogenies for h-type TRX proteins from Arabidopsis and TRX1 from Homo sapiens. Protein sequences from representative gene models were used with the MEGA 11 program with bootstrap test (1000 times) and neighbor-joining method. Branch lengths are proportional to phylogenetic distances.

Figure 3

Conserved features of Arabidopsis h-type TRXs and human TRX1 protein sequences. Highlighted in red are the active site Cys residues as well as the N-terminal Cys. The catalytic motif is underlined and additional C-terminal Cys residues unique to HsTRX1 are in blue. Glycine residues that undergo N-myristoylation are in green. Asterisks (*) denote conserved residues, (:) strong and (.) weakly similar amino acid properties.

Figure 4

Proposed TRX denitrosation mechanisms via the reductive (a) or transnitrosation pathway (b). In the reductive pathway (a), the nucleophilic Cys residue of the TRX displaces NO from the target Cys by heterolytic cleavage, resulting in the formation of an intermolecular disulfide bond between TRX and its target substrate. Subsequently, the resolving active site Cys attacks the mixed disulfide and gets oxidized, releasing the reduced substrate. Oxidized TRX is then recycled by NTR. (b) R-SNOs can undergo a transnitrosation reaction with another thiol leading to the transfer of NO.

Figure 5

Phylogenetic tree of Arabidopsis AKR proteins. The phylogenetic tree was constructed by searching for aldo-keto reductases (PTHR11732) in the PANTHER database and using the MEGA 11 program with bootstrap test (1000 times) and the neighbor-joining method. Highlighted in red are the four members of the subclass 4C, while marked in blue are AKR proteins that lack the Lys residue in their catalytical tetrad. Green denotes AKR proteins that show an N-terminal extension, indicating they may localize to chloroplasts or mitochondria. In addition to the AGI identifiers, other names given to each AKR are shown.

Figure 6

Multiple sequence alignment of AKRs from Arabidopsis. Red boxes highlight the catalytic tetrad residues, while green, cyan and red bars above the alignment denote the flexible loops defining the active site important for substrate specificity. Elements were assigned using the structural information of Arabidopsis AKR4C8 (PDB code 3h7r). Residues are color-coded based on their properties: red: positive; blue: hydrophobic; green: polar; orange: glycine; purple: negative; teal: aromatic; yellow: proline.

Figure 7

Direct (a) and indirect (b) denitrosation of proteins in plants. Enzymatic denitrosation systems have been reported in the literature. The thioredoxin system uses NADPH to remove the nitroso group from S-nitrosated target proteins via two distinct mechanisms. However, further studies have to show the contribution of the GRX and SRX-system in this process in plants. In contrast, the level of available NO is regulated by GSNOR and the newly identified AKR proteins, thereby modulating the S-nitrosation status of proteins indirectly. See text for further details.